DE69933131T2 - CELLS FOR THE PRODUCTION OF HELPER-DEPENDENT ADENOVIRUS VECTORS - Google Patents

Abstract

The present invention relates to cells for the production of helper dependent adenoviral vectors, including at least the following genic units: a first genic unit comprising an adenovirus defective genome having the inverted terminal repeats in head-to-tail configuration, the encapsidation signal inactivated, and at least one of the non-structural regions inactivated; a second genic unit comprising at least one inducible promoter and at least one of the regions inactivated in the first genic unit, said regions being under the control of said inducible promoter; whereby following the activation of the inducible promoter of the second genic unit and the infection of the cells with said helper dependent adenoviral vectors, the first genic unit and the second genic unit enable the production of said helper dependent adenoviral vectors in said cells in absence of helper vector.

Description

Territory of invention

The The present invention relates to the field of gene therapy, and more particularly the use and development of vectors of viral origin and of cell lines for their production.

background the invention

One important aspect in the development of gene therapy is the development of vectors, which introduce of genetic material in target cells. To be effective they have to be Vectors have several features: You must be able to large or including several transgenes Gene regulatory elements, absorb, but easily manipulated remain to enable their production on a pharmaceutical scale. About that In addition, it is essential that they are safe and of low toxicity, but the fortune for the introduction the transgenes in an efficient and selective manner into the target tissues preserved. After all Preferably, the vector should be given a suitable retention, expression and regulation of the transgene in the target cell.

Currently The adenovirus-based viral vectors seem to be those too be which for The most appropriate manipulations that enable them to to meet all these requirements. So far, they are becoming the most effective system for introducing heterologous Genes in mammalian cells, both in vivo and in cells cultured in vitro (Hitt, M.M., et al., 1997, Advances in Pharmacol., 40, 137-206).

This based on some interesting properties of adenoviruses (Ad), which is a DNA virus family (those who infect humans were in 57 serotypes) characterized by an icosahedral capsid, which lacks an outer shell, They are very infectious, but relatively harmless, infecting mainly Epithelial cells, can but also infect cells of other tissues, regardless of whether they are in the active replication phase. Because of your high infectivity In vitro, from cell lines of human origin, they can easily in large quantities getting produced. About that In addition, it has been proven that human DNA inserts by means of ad infection can be efficiently transferred into human epithelial cells (Horvitz, "Adenoviridae and their replication "in Virology, Field and Knipe, ed. Raven Press, NY; 1990; Pages 1679-1740).

Regarding the molecular biology of the virus has adenovirus, in particular the human adenovirus, a linear double-stranded DNA genome of about 36 kB, which is functional in two areas, non-structural and structural, is divided. The first region comprises regions which code for polypeptides, which are expressed in the first stages of infection, i. before viral DNA replication (E regions), the second includes Areas which for Encode polypeptides that are in the subsequent stages of the viral Cycle are expressed (L-range). After infection of a competent Cell, when the viral DNA reaches the nucleus, is the first to be transcribed Area of the E1a region, coding for proteins involved in transactivation the other areas, both E and L, are involved in the viral DNA are. The following transcribed E1b region codes for proteins encoding RNA synthesis, Both the viral and the host cell, regulate and protect the latter from the apoptotic effect that otherwise unfolded by E1a. That's why the E1a / E1b functions are for the viral replication essential.

Of the E2 area coded for Proteins that are directly involved in viral replication, such as. the viral DNA polymerase, the preterminal protein and proteins, which bind to the viral DNA. The E3 region codes for proteins, which for viral replication in cultured cells are not required, however, involved in the regulation of the antiviral immune response in vivo are. After all contains the E4 region groups of genes whose products affect gene expression reduce the host cell and transcribe the E2 and L regions of the viral Increase genome.

Of the L region of the viral genome essentially codes for proteins of the structural type or proteins, in any way involved in the assembly of viral particles.

In the first 40 years after the first isolation and characterization Ad viruses took the relevant modifications they made efficient carriers for the Transfer of genetic material.

• – das Vermögen des viralen Genoms zur Akzeptanz des Einbaus heterologer Gene zu erhöhen; und
• – intrazelluläre Toxizität, welche sich aus der Expression von Adenovirus-Genen ergibt, zu eliminieren.

In particular, the interventions on the ad genome were first performed to:

• To increase the ability of the viral genome to accept the incorporation of heterologous genes; and
• - to eliminate intracellular toxicity resulting from the expression of adenovirus genes.

Such Interventions mainly exist in the provision of progressive deletions of viral regions, whose functions are provided in trans.

In vectors of the first generation (deriv from human Ad serotypes 2 and 5), the deletion involved the E1 region, which renders the virus deficient in replicability if the proteins produced by such a transcriptional entity are not provided in trans.

A increase the size of the heterologous gene, which is to be inserted and the restriction of the multiplication of the recombinant Viruses in cell lines that complement such a defect, as they constitutively express genes of the viral E1 region was receive.

however the deletion of E1 is not sufficient per se for expression completely eliminate other genes of the E and L regions and vice versa to prevent the replication of viral DNA. It follows that in animals, infected with these vectors, the presence of viral Antigens and the onset of immune reactions, which are related to the destruction of are detected (Yang et al., Proc. Natl. Acad. Sci. 91: 4407-4411; 1994). This leads to the loss of the gene of therapeutic interest and the onset of inflammatory reactions. About that In addition, the persistence of an immunological memory of these reactions the effectiveness of a second administration of a Strongly reduce adenovirus vector of this type (Kozarsky et al., J. Biol. Chem. 269: 1-8; 1994).

Consequently were new adenoviral vectors of the second generation, which carry different combinations of deletions of early genes, constructed, to improve the safety profile of adenovirus vectors. Of vectors that combine different E1, E2, E3 and / or E4 deletions, has been demonstrated to be less cytotoxic in vitro and more stable in the mouse liver than the classical first generation ΔE1 vectors (Gao, G-P., 1996, J. Virol. 70: 8934-8943; Dedieu, J-F., 1997, J. Virol. 71: 4626-4637; Gorziglia, M.I., 1996, J. Virol. 70: 4173-4178; Amalfitano, A., 1998, J. Virol. 72: 926-933). in vifro experiments demonstrated that such deletions effectively reduced toxicity, but not eliminated.

Furthermore were vectors that extra Deletions contributed to the capacity of the viral genome Acceptance of the insertion of heterologous genes further increased (Englehardt et al., Proc. Natl. Acad Sci. 91: 6196-6200; 1994; Bett et al., Proc. Natl. Acad. Sci. 91: 8802-8806; 1994; Yeh, P., et al. 1996, J. Virol. 70: 559-565). However, the maximum capacity exceeded one First generation adenovirus vector not 8 kb, whereas those of a ΔE1 / E3 / E4 vector Reached 11 kb and the foreign genes equally in the range E1 or E3 can be inserted.

In This connection was after the discovery that a minimal Share, about 600 bp, of viral DNA strictly for replication and packaging recombinant vectors is required, completely deficient adenovirus vectors, consequently complete from the presence of helper viruses for replication and theirs Depend on assembly into viral particles.

such helper-dependent Adenovirus vectors (AdHD) carry minimal Ad sequences, which the for the replication and packaging contain sufficient signals. All other factors which for The virion production required are in trans from one Helper virus provided. The helper genome is in such a Way constructed, that its sequences, the packaging signals contained in vivo through the use of specific recombination systems, such as. the cre / lox system (WO97 / 32481) can be easily eliminated can.

• – Zelllinien, welche transformiert sind, um sie zur Expression der Gene, welche für die Gruppe von Adenovirus-E1-Proteinen und eine Rekombinase, gewöhnlich "cre", codieren, zu befähigen;
• – einem Helfer-Adenovirus, worin der E1-Bereich deletiert ist und worin die viralen DNA-Sequenzen, welche für dessen Verpackung im Inneren des reifen Virions erforderlich sind, von Rekombinationsstellen flankiert werden, welche als Substrate für die Rekombinase ("IoxP" im Falle von "cre") fungieren;
• – dem adenoHD-Vektor, welcher das Transgen von Interesse trägt.

Accordingly, the relevant strategies for the production of helper-dependent adenovirus virions (adenoHD) are based essentially on the use of three elements:

• Cell lines transformed to enable them to express the genes encoding the group of adenovirus E1 proteins and a recombinase, usually "cre";
• A helper adenovirus, wherein the E1 region is deleted, and wherein the viral DNA sequences required for its packaging inside the mature virion are flanked by recombination sites which serve as substrates for the recombinase ("IoxP" in the case of from "cre");
• The adenoHD vector carrying the transgene of interest.

Even though this strategy is a notable improvement over the represents strategies used in the vectors of the first generation still brings some problems with it. For a production in the pharmaceutical Scale brings the requirement of controlling three independent components (the cell line, the helper adenovirus and the transgenic vector), which are difficult to overcome are, and unacceptable production costs.

First, the cre-type recombinases catalyze both the deletion and insertion of the DNA regions flanked by the IoxP sites. The cleavage reaction is usually up to 20-fold more efficient than the opposite, but complete removal of the helper from viral production can never be achieved. This is unacceptable especially in pharmaceutical practice and a helper contamination of Präpa Therapeutic use is a very serious problem.

A second restriction This strategy type is based on the difficulty, the quantitative relationships between helper virus and helper-dependent virus during the To optimize the amplification process. Thus, the vector amplification achieves rarely the efficiency of the first generation vectors and often yields of up to an order of magnitude less are obtained. As in the previous case, this problem is dramatic when one Production on an industrial scale is necessary and the costs become practically unsustainable.

to solution of the problem resulting from the contamination with helper virus and reducing the number of components is an alternative Strategy known in the art, that cell lines to manipulate them to express all the factors involved in the packaging the defective virus are needed to enable so as to eliminate the helper virus.

in the Prior art there are several descriptions of cells which express one or more viral protein (s) (see for example WO98 / 13499). Such cell lines can used to produce adenovirus vectors to which the complementary missing viral proteins. However, according to this strategy, it is extremely difficult the exact coordination of events between viral DNA replication and to achieve the expression of the structural proteins, so that the natural infection leads to the massive production of virus particles, which are responsible for the lytic Phase is typical. Accordingly, with adoption of these strategies the viral cell cycle can not be mimicked. In his absence Only a limited yield capacity can be achieved.

Summary the invention

task The present invention is a helper cell line, which the Production of helper-dependent Adenovirus vectors in the complete absence of any Type of helper virus allows. Such a cell line was indeed constructed to be all essential Functions for the ad virus replication are contained in the cells, in particular split into two genetic units, one strictly regulated Expression allowed.

The first genetic unit comprises at least one defective adenovirus genome, in which the inverted terminal repeats (ITRs) are in a head-to-tail configuration present and the packaging signal and at least one of the non-structural Areas is disabled.

Such non-structural areas can be expressed Be areas or cis-acting elements. The expressed areas can both non-translated non-structural genes, e.g. the VA genes, or translated non-structural genes such as the E1, E2 and E4 genes.

In a preferred embodiment the inactivation is carried out by complete or, more preferably, by partial deletion of at least one such region; the packaging signal is also preferably by full or partial deletion inactivated.

A such first genetic entity may be on an episomal unit be localized or integrated into the genome of the host cells.

in the In the first case, the episomal unit must comprise at least one element, which replicates the episomal unit itself with a low copy number allows. Such an element may consist of sequences involving DNA replication and their retention in the core, be built (Calos, M. P., 1996, Trends Genet. 12: 463-466) or from the replication origin the origin of the replication system of a virus activated by at least an activation factor.

Of the Replication origin must in any case be on the episomal unit be localized; the activation factor may instead be in the Cell introduced or the relevant gene may be on the episomal unit, including the first genetic unit, (or in any other replicating unit within the cell) or can be located in the genome of the Host cell be integrated. In any case, the gene should be for the Akti vierungsfaktor within the cells of the present invention encoded, expressed to replicate the first genetic To allow unity in the episomal unit.

One Example of an origin of a replication system used for the present Invention is functional, is the relevant system of Epstein-Barr virus (which is the preferred), wherein the origin of replication is the OriP element and the activation factor is the EBNA-1 protein. however are different systems, e.g. that of bovine papillomavirus (BPV) (Calos, M.P. (1998), Proc Natl Acad Sci., USA 95: 4084), or those from vectors based on an SV40 origin T-antigen system, also suitable.

The presence of such origin of a replication system allows the first geneti low copy number replication, whereas the ITRs in the first genome allow replication in a high copy number in the presence of the proteins encoded by the E2 regions.

If instead, the first genetic unit in the genome of the host cells integrated ITR sequences in a head-to-tail configuration at both ends the first genetic unit. Such ITRs allow in indeed the release and replication of the first genetic unit in high copy number in the presence of the regulatory proteins, which are encoded by the above genes of the E2 regions.

In any case can furthermore, regulatory elements which inhibit the expression of at least one of the non-structural areas in the first genetic Control unit, contained in such a first genetic unit be or can also in the cell, preferably integrated in the cell genome.

One Examples of such regulatory elements are the elements which by Rittner (Rittner, K., et al., (1997) J. Virol 71: 3307-3311) have been described.

The second genetic unit comprises an inducible transcription unit, which are the viral non-structural regions present in the defective ad genome the first genetic unit are inactivated, under control an inducible promoter responsive to strict regulation includes. Such areas can for example, be E1, E2A, E2B and / or E4.

One inducible promoter in the genetic units of the present Invention and in particular in the second genetic unit a promoter induced by an inducer. An example is the tetracycline promoter, which is the preferred, and promoters, which is due to the presence of other inducers such as ecdysone, rapamycin, RU486, dexamethasone or a heavy metal such as Zn or Cd regulated be, are alike suitable.

One such promoter can be functional with regulatory elements, e.g. responsive to tetracycline Transactivators and / or silencers (rtTA and tTS), both enable the strict regulation of the transcription unit, be linked.

The second genetic unit can enter the host cell through a vector introduced which may be a virus or plasmid into the genome of the Host cell integrated or present on an episomal unit can; In any case, she must during the growth phase of the cell line are kept inactive to the to avoid cytotoxic effect of viral proteins. To this Purpose can be specific regulatory elements affecting the specific repression of the transcription unit enable, to be included in the second genetic unit. An example such a repressor is the Tet / krab fusion protein.

Such Repressor elements are in each case functional with the inducible Promoter and the regulatory element expressing the expression the transcription unit described above, coordinated.

In Absence of the inducer, indicating the relevant inducible Promoter of the second genetic unit (transcription unit) the non-structural areas contained in it indeed not expressed, the first genetic unit is inactive and thus There is no production of viral protein, which is responsible for the cell is toxic.

To the Start production of helper-dependent adenovirus vector infecting the helper cell line with the helper-dependent adenovirus vector, in big Quantities to be produced, and in the meantime (however also before or after infection with the defective adenovirus vector) the inducer of the inducible promoters of the second genetic Unit present in the cell added or their expression induced within the cell.

The Result of such an action will be the production of the adenoviral Proteins from the early Areas included in the second genetic unit are, be coded, what the activation of the transcriptional Cascade of adenoviruses implicated in ITR-mediated replication associated with the first genetic unit.

The The consequence of this coordinated series of events is the accumulation of large quantities Structural proteins of the virus, similar the one who during the natural one Infection occurs. The viral proteins are used in the construction of the viral particle and thus lead to efficient Production of the helper-dependent viral vector.

In this regard, it should be emphasized that for the purposes of the present invention, a helper-dependent adenovirus vector is an adenovirus vector whose viral cycle can not be completed in the absence of a helper, particularly an adenovirus vector in which the viral genome is complete or partially deleted, with Exception of the packaging signal and the inverted terminal repeats (ITRs). For the purposes of the present invention they are referred to hereafter and previously as helper-dependent adenovirus or helper-dependent adenovirus virions.

in the Essentially, this strategy aims to replicate the phenomenon of viral latency; in the helper cell, a viral genome gets through Suppression of expression of genes encoding the adenovirus transcription cascade underlying, held in a latent phase. At the time infection / transformation with the defective adenovirus vector that produces is to be activated, the lytic phase is activated by induction of Expression of Proteins Encoded by the Early Genes from the ad genome, which is included in the first genetic unit, deleted and under the strict transcriptional control in the second genetic Unit were made.

The previously latent adenovirus genome present in the first genetic unit is therefore involved in active replication and Transcription, but only the helper-dependent vector that produces is to be packed, since only he is a functional packaging signal has.

As a consequence of the fact that the system object of the present invention includes two instead of three elements, the production of the helper-dependent adenovirus vector is greatly facilitated, which after a large scale preparation yields a final yield of titer levels between 10 ⁹ and 10 ¹² particles / milliliter allowed.

A such property of the cell lines of the present invention particularly relevant when used in large-scale productions such as those used in the pharmaceutical industry. Accordingly the present invention allows for improved production of the vectors, which therapeutic genes suitable for the treatment of human or veterinary diseases.

In In any case, it should be noted that the two genetic units can be introduced into the cell at different times. Accordingly Cell lines are disclosed which are at least only the first genetic Contain unit, and cell lines, which are at least only the second contain genetic unit. The second genetic unit in the first Case and the first genetic unit in the second case can be found in indeed before or simultaneously with the transfection of the cell the helper-dependent Added adenovirus vector become.

• – eine erste genetische Einheit, umfassend ein defektes Adenovirus-Genom, bei dem die invertierten terminalen Repeats in einer Kopf-zu-Schwanz-Konfiguration vorliegen und bei dem sowohl das Verpackungssignal als auch mindestens einer der nicht-strukturellen Bereiche inaktiviert sind;
• – eine zweite genetische Einheit, umfassend mindestens einen induzierbaren Promotor und mindestens einen der in der ersten genetischen Einheit inaktivierten Bereiche, wobei die Bereiche unter der Kontrolle des induzierbaren Promotors stehen;

wodurch nach der Aktivierung des induzierbaren Promotors der zweiten genetischen Einheit und der Infektion der Zellen mit den helfer-abhängigen Adenovirus-Vektoren die erste genetische Einheit und die zweite genetische Einheit die Produktion der helfer-abhängigen Adenovirus-Vektoren in den Zellen in Abwesenheit von Helfervirus ermöglichen.Therefore, in connection with the subject matter of the present invention disclosed above and below, the cells suitable for the production of helper-dependent adenovirus vectors are characterized by including at least one of the following genetic units:

• A first genetic unit comprising a defective adenovirus genome in which the inverted terminal repeats are in a head-to-tail configuration and in which both the packaging signal and at least one of the non-structural regions are inactivated;
• A second genetic unit comprising at least one inducible promoter and at least one of the regions inactivated in the first genetic unit, said regions being under the control of the inducible promoter;

whereby after activation of the inducible promoter of the second genetic unit and infection of the cells with the helper-dependent adenovirus vectors, the first genetic unit and the second genetic unit enable production of the helper-dependent adenovirus vectors in the cells in the absence of helper virus ,

Further objects The present invention relates to the cells described above, wherein the first genetic unit is integrated into the genome and the ITRs present at both ends of the first genetic unit in a head-tail configuration; the cells described above, wherein the first genetic unit enclosed in an episomal unit that is an element which involves the replication of the genetic unit in one low copy number allows. In the latter case, special cases are cells in which the element, which allows the replication of the episomal unit, the Replication origin of the replication system origin Virus is or is derived from it; the case where the relevant Activation factor from outside the cell is introduced into the cell becomes; the cases in which the gene, which for encodes the activation factor located on the episomal unit or in another transcriptional unit within the cell or alternatively integrated into the genome of the host cell.

Of the The origin of the replication system described above may be that of Epstein-Barr virus (EBV) and the origin of the replication element is OriP and the activation factor is EBNA-1.

Other specific cases with respect to each case above are the following: the case where the packaging signal and / or the non-structural Areas of the first genetic unit are completely or partially deleted; the case where the regions inactivated in the first genetic entity and present in the second genetic entity represent at least one of the early regions consisting of the group consisting of E1, E2A, E2B and E4 regions More specifically, the case where the regions are E1 and E4 are the case where the regions are E1, E4 and E2A, the case where the regions are E1, E4 and E2B polymerase, and the case in which are the regions E1, E4 and preterminal E2B protein (PTP).

Further Cases are those in which the viral areas, which in the first genetic unit, functional with at least one regulatory Linked item which allows the strict control of their expression; Cells in which the promoter on the second genetic Unit specifically for the tetracycline operator, the promoters of which Steroid hormone receptors are regulated, the promoter from the ecdysone receptor regulated by rapamycin Promoter, RU486 regulated promoter and metal ions regulated metallothionein promoter is; in these cases will be the relevant inducers of tetracycline, ecdysone, rapamycin, dexamethasone RU486 or a heavy metal such as Zn or Cd formed; Cells, in which the viral areas in the second genetic unit are functional with Linked elements which are the expression of the non-structural Control areas, and in particular the case in which such elements Tetracycline-responsive transactivators and / or silencers are; the cells in which at least one of the non-structural Areas in the second genetic unit complete or in part from an adenovirus genome, preferably human adenovirus, in particular Adenoviruses of serotypes AD2 or AD5. In these latter cases can the different sections of the non-structural areas, present on the second genetic unit, of adenoviruses of the same serotype or of adenoviruses of a different one Serotype originate.

Further Cases of Interest are the cells in which the adenovirus genome of the first complete genetic unit or is derived in part from the adenovirus genome of mammalian adenoviruses, preferably Human adenoviruses, preferably adenovirus of the serotype AD2 and AD5. In these latter cases can the different sections of the adenovirus genome on of the first genetic unit, of adenoviruses of the same Serotype or from adenoviruses of a different serotype.

The Genetic units of the present invention can be used in at least one vector, preferably a plasmid, which in held in the episomal phase or not, are introduced into the cells; both in the case where they are integrated into the host genome, as well as in the case where they are on a replicating unit within the cell such first and second genetic units are functional with genomic sequences encoding the expression of the functions are coded by at least one of the units, in regulated Make sure you're linked be.

Further can all cells described above are eukaryotic cells, preferably Mammalian cells, preferably human cells, and in this latter case also preferably Cells that allow adenovirus replication. Especially is the cell line A549 (human lung carcinoma ATCC CLL 185) for the derivative the cells of the present invention.

• a) Einführen der ersten genetischen Einheit wie oben beschrieben in eine Zelllinie;
• b) Einführen der zweiten genetischen Einheit wie oben beschrieben in die Zelllinie von Schritt a).

Also disclosed is a method of producing the above-mentioned cells comprising the present steps:

• a) introducing the first genetic unit into a cell line as described above;
• b) introducing the second genetic unit into the cell line of step a) as described above.

Specific Cases become represents by such a procedure, in which the first genetic unit of step a) and the second genetic unit of step b) in a plasmid or virus vector. Especially regarding the Plasmid vectors, which for the present invention are suitable, come cloning vectors such as pUC, pBluescript and pLitmus into consideration.

specially in terms of of the viral vectors, which for the present invention are suitable, there are retroviral vectors, adeno-associated viral vectors, herpes simplex vectors and from Epstein-Barr virus derived vectors.

Also such methods can preferably be carried out with a mammalian eukaryotic cell line, preferably with a human cell line and preferably with the cell line A549 (human lung carcinoma ATCC CLL 185).

Also discloses the use of the above cells for production of helper-dependent adenoviral vectors or the cell line A549 (human lung carcinoma ATCC CLL 185), both in vivo and in vitro.

Also disclosed is the use of the above cells to produce the helferab pending adenovirus vectors according to a technique known in the art.

Such helper dependent Adenovirus vectors should contain at least one gene, which preferably may be a gene of therapeutic interest, its products especially for the gene therapy are useful, especially directed against human or veterinary Diseases. Special cases are the following where the helper-dependent adenovirus vectors at least one gene of interest in genetic engineering or in methods of producing transgenic animals; of the Case in which the products of the activity of the gene peptides or ribozymes are.

One Another object of the present invention are compositions, which comprise the previously described cell and a vehicle or a carrier, characterized in that the composition is 99% to 100% is free of helper viruses.

compositions which a vehicle or a carrier and one of the cells described above, wherein the cells the first and second genetic units and the helper-dependent adenovirus vector are included, are an object of the present invention. Such a vehicle or such a carrier may be water, glycerin, ethanol, saline or the like or combinations be of it.

Especially relevant cases are those in which the vehicle or vehicle in all these compositions with the active principle of action, which the above helper-dependent Adenovirus vector and one of the cells described above, is pharmaceutically acceptable and compatible. Pharmaceutically acceptable carrier are well known in the art, i. sterile aqueous solutions, which may contain the active principle or also buffers, such as e.g. Sodium phosphate at a physiological pH and / or a physiological saline solution, such as. phosphate buffered saline. Furthermore can other excipients, e.g. a wetting agent or emulsifier, promoting solution Agents, pH buffering agents, stabilizers and colorants or the like or any other known in the art Additive to be included in such compositions.

In In this context, pharmaceutically acceptable or compatible carriers or Vehicle to the materials known in the art for administration to or on an individual, for example a mammal, especially a human, without causing an undesirable physiological effect to be suitable. Examples of such effects are nausea, Upset stomach, dizziness and the like.

The Preparation of such compositions, which pharmaceutical or may be pharmacological compositions in which the active Action principles solved or dispersed, is well known in the art. In general such compositions are for parenteral administration or in any case as injectable compositions, either as liquid solutions or suspension. In any case, you can solid forms, suitable for solutions or suspension in liquid before use. Preparations for others desired Routes of administration according to Stand techniques well known in the art, e.g. the oral route, dermal plasters, suppositories or also an emulsified preparation included in the present invention.

Specific Ask cases the compositions containing the helper-dependent adenovirus vector or at least one of the cells of the present invention, the helper-dependent adenovirus vectors lock in, containing at least one gene suitable for molecules of therapeutic interest encoded, molecules, which may also be peptides or ribozymes, in particular of use in gene therapy, more specifically directed against human diseases.

Also discloses the use of the cells of the present invention as a medicament and in particular for the production of the abovementioned Compositions, in particular for pharmaceutical or pharmacological Compositions suitable for the gene therapy are suitable.

Also discloses the process for producing at least one of compositions described above, which are pharmaceutical compositions or material compositions.

• – eine Zusammensetzung, umfassend mindestens eine oder oben beschriebenen Zellen, einschließlich einer ersten genetischen Einheit, einer zweiten genetischen Einheit und eines helfer-abhängigen Adenovirus-Vektors;
• – eine Zusammensetzung, umfassend einen Induktor, welcher zur Aktivierung des induzierbaren Promotors in der zweiten genetischen Einheit in der Lage ist.

Also disclosed are kits comprising at least one of the above-described compositions for the production of the helper-dependent adenovirus vectors of the present invention in vitro, in vivo or ex vivo. Of special interest is the kit, which comprises:

• A composition comprising at least one or above-described cells, including a first genetic unit, a second genetic unit and a helper-dependent adenovirus vector;
• A composition comprising an inducer which is capable of activating the inducible promoter in the second genetic unit.

• – eine Zusammensetzung, umfassend mindestens eine der oben beschriebenen Zellen, einschließlich einer ersten genetischen Einheit und einer zweiten genetischen Einheit;
• – eine Zusammensetzung, umfassend mindestens einen helfer-abhängigen Adenovirus-Vektor, einschließlich eines heterologen Gens, insbesondere von therapeutischem Interesse; und gegebenenfalls
• – eine Zusammensetzung, umfassend einen Induktor, welcher zur Aktivierung des induzierbaren Promotors in der zweiten genetischen Einheit in der Lage ist.

Of particular interest is also the case where the kit includes:

• A composition comprising at least one of the cells described above, including a first genetic unit and a second genetic unit;
• A composition comprising at least one helper-dependent adenovirus vector, including a heterologous gene, in particular of therapeutic interest; and optionally
• A composition comprising an inducer which is capable of activating the inducible promoter in the second genetic unit.

The Kits disclosed herein may a kit for the therapeutic use (production in vivo or ex vivo of the helper-dependent adenovirus vector, the at least one gene of therapeutic interest, in particular in gene therapy) and a kit for in vitro production a helper-dependent Adenovirus vector containing at least one gene, preferably of therapeutic Interest, contains, be.

The The invention will be described in more detail with the aid of the appended figures become.

Description of the figures

• A) Karte des AD5-Shuttle-Vektors (erste genetische Einheit): Ad5ΔΨΔE1/E4 repräsentiert das Genom eines humanen Adenovirus, vollständig mit Ausnahme der Deletion der E1-, E4-Bereiche und desjenigen, der das Verpackungssignal einschließt (Ψ); AdITRs repräsentieren die Ad5-ITR-Gensequenzen in Kopf-Schwanz-Konfiguration; Hygro^r repräsentiert das Hygromycinresistenzgen; Amp^r das Ampicillinresistenzgen; EBNA-1 den viralen Transaktivator des Epstein-Barr-Virus (EBV) und OriP repräsentiert den Ursprung der latenten Replikation des EBV-Virus.
• B) Karte des E1/E4-Vektors (zweite genetische Einheit): wobei E1 und E4 DNA-Sequenzen repräsentieren, die den E1- und E4-Adenovirus-Bereichen entsprechen, MSC I und II Sequenzen sind, welche Mehrfachklonierungsstellen einschließen; PminCMV die minimalen Promotoren des Cytomegalovirus (CMV) sind, TRE (Tetracyclin-Response-Element) sieben wiederholte Sequenzen des Operators des E. coli-Tetracyclinresistenzgens repräsentiert; β-Globin-polyA die Polyadenylierungsstelle des β-Globins ist; ColE1ori der Replikationsursprung von E. coli ist; SV40-polyA die SV40-Virus-Polyadenylierungsstelle ist.
• C) Karte des pTet-On/Off-Vektors (regulatorisches Element): wobei Pcmv und Psv40 die Promotoren von CMV bzw. SV40 sind, (r)tTa den steuerbaren Transaktivator von Tetracyclin (oder den invertierten, d.h., denjenigen, der nur von einem Tetracyclin aktiviert wird) anzeigt. Dieser Transaktivator besteht wiederum aus dem Gen, welches für den Tetracyclin-Repressor (oder dessen Mutante rtetR der entgegengesetzten Funktion) in Fusion mit der VP16AD-Domäne des Herpes-simplex-Virus codiert. Neo^r repräsentiert das Neomycinresistenzgen.
• D) Karte des pTet-KRAB-Vektors (regulatorisches Element): im wesentlichen ähnlich dem pTet-On/Off-Vektor, wobei die für die VP16AD-Domäne des Herpes-simplex-Virus codierenden Sequenzen durch diejenigen der KRAB-Domäne des humanen Kox-Gens ersetzt sind.

1 shows in the four fields the schematic maps of vectors useful for deriving the helper cell lines of the present invention:

• A) Map of the AD5 shuttle vector (first genetic unit): Ad5ΔΨΔE1 / E4 represents the genome of a human adenovirus, with the exception of the deletion of the E1, E4 regions and the one that includes the packaging signal (Ψ); AdITRs represent the Ad5 ITR gene sequences in the head-tail configuration; Hygro ^r represents the hygromycin resistance gene; Amp ^r the ampicillin resistance gene; EBNA-1 is the viral transactivator of Epstein-Barr virus (EBV) and OriP represents the origin of latent replication of the EBV virus.
• B) Map of the E1 / E4 vector (second genetic unit): wherein E1 and E4 represent DNA sequences corresponding to the E1 and E4 adenovirus regions, MSC I and II are sequences which include multiple cloning sites; PminCMV are the minimal promoters of cytomegalovirus (CMV), TRE (tetracycline response element) represents seven repeated sequences of the operator of the E. coli tetracycline resistance gene; β-globin-polyA is the polyadenylation site of β-globin; ColE1ori is the replication origin of E. coli; SV40 polyA is the SV40 virus polyadenylation site.
• C) map of the pTet-on / off vector (regulatory element): where Pcmv and Psv40 are the promoters of CMV and SV40, respectively (r) tTa the controllable transactivator of tetracycline (or the inverted, ie, the only of a tetracycline is activated). This transactivator again consists of the gene encoding the tetracycline repressor (or its mutant rtetR of opposite function) in fusion with the VP16AD domain of the herpes simplex virus. Neo ^r represents the neomycin resistance gene.
• D) Map of the pTet-KRAB vector (regulatory element): substantially similar to the pTet-on / off vector, wherein the sequences coding for the VP16AD domain of the herpes simplex virus are characterized by those of the KRAB domain of the human Kox Gene are replaced.

• A) Karte des AD/EBV-Shuttle-Vektors pSA-1 (erste genetische Einheit): Ad5 repräsentiert das Genom eines humanen Adenovirus, vollständig mit Ausnahme der Deletion der E1-, E4-Bereiche und desjenigen, der das Verpackungssignal einschließt (Ψ); AdITRs repräsentieren die Ad5-ITR-Gensequenzen in Kopf-Schwanz-Konfiguration: Hygro^r repräsentiert das Hygromycinresistenzgen; Amp^r repräsentiert das Ampicillinresistenzgen; OriP repräsentiert den Ursprung der latenten Replikation des EBV-Virus; Tet-Eptamer repräsentiert ein Eptamer von Tetracyclin-Repressor-DNA-Bindungsstellen. Die DNA-Sequenzen, welche deletiert wurden, um eine unterschiedliche ΔE2-Version dieses Vektors zu erzeugen, sind ebenfalls angegeben: DBP-Deletion repräsentiert die Deletion der gesamten codierenden Sequenz des DNA-bindenden Proteins; Polymerase-Deletion repräsentiert die Deletion von 608 bp innerhalb des Aminoterminus des Polymerasegens; pTP-Deletion repräsentiert die Deletion von zwei DNA-Segmenten innerhalb des Hauptexons des Gens des präterminalen Proteins.
• B) Karte des AD/EBV-Shuttle-Vektors pSA-2 (erste genetische Einheit), das einzige Element, welches pSA-2 von pSA-1 unterscheidet, ist die Insertion des EBNA-1-Gens neben dem OriP-Replikationsursprung.
• C) Karte von pIREStTS/rtTApuro (regulatorisches Element): wobei CMV-IE den "immediateearly"-Promotor des Human-Cytomegalovirus repräsentiert; rtTA DNA-Sequenzen repräsentiert, die dem reversen Tetracyclin-Transaktivator entsprechen; IRES die Sequenz ist, welche die interne Ribosomeneintrittsstelle einschließt; tTS-Kid die Sequenz repräsentiert, welche der Fusion zwischen dem tet-Repressor und einer Silencer-Domäne von Kid-1 entspricht; Kid-1-S-Domäne die Silencer-Domäne von Kid-1 ist; SV40-PuroR-polyA die Puromycinresistenzgen-Expressionskassette repräsentiert; Amp das Ampicillinresistenzgen ist.

2 Figure 12 shows the schematic maps of vectors useful for deriving the helper cell lines of the present invention:

• A) Map of the AD / EBV Shuttle Vector pSA-1 (First Genetic Unit): Ad5 represents the human adenovirus genome, with the exception of the deletion of the E1, E4 regions and the one including the packaging signal (Ψ) ; AdITRs represent the Ad5 ITR gene sequences in head-tail configuration: Hygro ^r represents the hygromycin resistance gene; Amp ^r represents the ampicillin resistance gene; OriP represents the origin of latent replication of the EBV virus; Tet-Eptamer represents an eptamer of tetracycline repressor DNA binding sites. The DNA sequences which have been deleted to produce a different ΔE2 version of this vector are also indicated: DBP deletion represents the deletion of the entire coding sequence of the DNA-binding protein; Polymerase deletion represents the deletion of 608 bp within the amino terminus of the polymerase gene; pTP deletion represents the deletion of two DNA segments within the major exon of the protein of the pre-terminal protein.
• B) Map of the AD / EBV shuttle vector pSA-2 (first genetic unit), the only element that distinguishes pSA-2 from pSA-1, is the insertion of the EBNA-1 gene next to the OriP origin of replication.
• C) Map of pIREStts / rtTApuro (regulatory element): where CMV-IE represents the "immediateearly" promoter of human cytomegalovirus; rtTA represents DNA sequences corresponding to the reverse tetracycline transactivator; IRES is the sequence that includes the internal ribosome entry site; tTS-Kid represents the sequence corresponding to the fusion between the tet repressor and a silencer domain of Kid-1; Kid-1-S domain the Silen cer domain of Kid-1; SV40 PuroR polyA represents the puromycin resistance gene expression cassette; Amp is the ampicillin resistance gene.

detailed Description of the invention

Disclosed is the construction and use of the first genetic unit as described in the summary of the invention for the derivation the helper cells of the present invention.

The first genetic unit can be applied as a plasmid vector of standard recombinant DNA techniques. A modification (Insertions, deletions, mutation) of the first genetic unit can be prepared according to the method described by A.F. Stuart and coworkers (Zhang et al., Nat. Genet. 1998; 20: 123-126).

• i) ein Adenovirus-Genom, vollständig mit Ausnahme der Deletion von mindestens einem der nicht-strukturellen Bereiche, welche für den Ablauf der Adenovirus-Transkriptionskaskade essentiell sind, und desjenigen, der das Verpackungssignal einschließt;
• ii) zwei Sequenzen von invertierten terminalen Repeats von Ad (ITRs) in einer Kopf-zu-Schwanz-Konfiguration, welche das Adenovirus-Genom flankieren; und im Allgemeinen ist auch
• iii) ein Selektionsmarker, wie z.B. ein G418-Resistenzgen, Hygromycin B-Resistenzgen, Puromycinresistenzgen, Bleomycinresistenzgen, vorhanden, der zur Selektion einer Zelllinie, welche die Einheit stabil aufrechterhält, verwendet werden kann.

The first genetic unit, when integrated into the genome of the host cell, typically includes:

• i) an adenovirus genome, with the sole exception of the deletion of at least one of the non-structural regions essential for the course of the adenovirus transcription cascade and the one involving the packaging signal;
• ii) two sequences of inverted terminal repeats of Ad (ITRs) in a head-to-tail configuration flanking the adenovirus genome; and in general, too
• iii) a selection marker such as a G418 resistance gene, hygromycin B resistance gene, puromycin resistance gene, bleomycin resistance gene present, which can be used to select a cell line which stably maintains the unit.

Cell lines which integrates the first genetic unit into the host chromosome can contain by transforming the cell with the vector DNA using of standard DNA transfection methods and culturing the transformed cells in a selective manner Medium (+ G418 or hygromycin B or bleomycin or puromycin) obtained by the techniques known in the art.

The Transcription and replication of an adenovirus vector inserted into the Host chromosome between two ITR junctions in a head-to-tail configuration can also be activated by providing deleted viral factors and / or viral functions, the in the adenovirus genome were inactivated in trans DNA transfection or infection with viral vectors or direct introduction active viral polypeptides.

• iv) den Ursprung der latenten Replikation eines Virus und insbesondere des Epstein-Barr-Virus (EBV);
• v) die invertierten terminalen Repeats (ITRs) von Ad-Gensequenzen in einer Kopf-Schwanz-Konfiguration.

In the case where the first genetic unit is instead held on an episomal unit, the vector used for introduction of the first genetic unit has been termed a "shuttle vector", such shuttle vector being a vector (plasmid or virus ) is in an episomal form and has been constructed to include the elements specified in item i) above, generally the elements given in item iii) above, and further comprising:

• iv) the origin of latent replication of a virus, and in particular Epstein-Barr virus (EBV);
• v) the inverted terminal repeats (ITRs) of Ad gene sequences in a head-to-tail configuration.

Of the Vector in this case was due to the characterizing presence the EBV elements referred to as "Ad / EBV shuttle vector". In the Act is also for including the gene encoding EBNA-1 protein.

In a preferred embodiment of such a vector are the E1 and E4 genes in the adenovirus genome of item i) deleted and the basic activity of the E2 promoter is by insertion of a Tet-Silencer binding site into the viral chromosome upstream of the E2 promoter. In a second preferred embodiment of such a vector are the E1 and E4 genes in the adenovirus genome and deleted E2A genes. In a third preferred embodiment of such a vector in the adenovirus genome are the E1 and E4 genes and E2B genes deleted.

One The essential characteristic of such a "shuttle vector" is its ability to replicate a high copy number per cell reproduced as soon as the transcription the ad genes were activated. This is made possible by the presence the inverted terminal repeats (ITRs) of adenovirus in one Head-tail configuration in the construct.

In a preferred embodiment of such a vector, the Ad genome is that of the serotype Ad5, in a second preferred embodiment, such comprises Vector the genome of the serotype Ad2. Combinations with other serotype genomes or any other member of the Adenoviridae family possible and could be preferred in some applications.

In a preferred embodiment of such a vector, the ITRs are those of the serotype Ad5. In a further preferred embodiment In this form of vector, the ITRs are those of the serotype Ad2. Combinations with ITRs of other serotypes or any other member of the Adenoviridae family are possible and may be preferred in some embodiments.

eukaryotic Cells can with the first genetic unit after transfection protocols like the calcium phosphate method or lipofection or using be transformed by DNA microinjection. Cells, which the first contain genetic unit, taking advantage of the presence of a selection marker on the episome by adding the appropriate Antibiotics are selected to the culture medium. permissive Cells containing the first genetic unit in episomal form, can in the presence of the viral functions present in the defective Ad genome present in the first genetic unit is present, deleted or inactivated were to promote the propagation of an adenovirus.

A cell line obtained as described above can be used for the insertion of the second genetic unit, in which in the first genetic unit inactivated adenovirus functions under a strict transcriptional Control are used.

The second genetic unit can be recombinant by standard methods DNA techniques, e.g. direct cloning of DNA fragments obtained by DNA digestion with restriction enzymes, in vectors, PCR or by homologous recombination technique in E. coli or eukaryotic Cells are obtained.

• I) die Nicht-Strukturbereiche, die in dem Adenovirus-Genom der ersten genetischen Einheit inaktiviert wurden, integriert in das Genom der Wirtszelle oder in einem "Shuttle-Vektor" vorliegend;
• II) einen induzierbaren Promotor, der streng regulierbar sein kann; und gegebenenfalls
• III) zwei Dimere von Isolatorsequenzen, welche die Transkriptionseinheit flankieren;
• IV) regulatorische Elemente, wie z.B. Tetracyclin-Response-Elemente.

The relevant vectors used to introduce the second genetic unit accordingly contain as characterizing elements:

• I) the non-structural regions inactivated in the adenovirus genome of the first genetic unit integrated into the genome of the host cell or in a "shuttle vector";
• II) an inducible promoter which may be strictly regulatable; and optionally
• III) two dimers of insulator sequences flanking the transcriptional unit;
• IV) regulatory elements, such as tetracycline response elements.

One The essential characteristic of such a vector is the ability to acquire during the Growth step of the cell line to be kept strictly inactive, to avoid the cytotoxic effect of viral proteins.

In a preferred embodiment of such a vector, the Ad regions are those of the serotype Ad5, in a second preferred embodiment of such a vector Ad areas are those of serotype Ad2. combinations with ad ranges of other serotypes or any other member The Adenoviridae family are possible and could be preferred in some applications.

In a preferred embodiment of such a vector, the inducible promoter consists of a Element that responds to tetracycline. The regulation of expression based in this case on the use of the two regulatory Elements of tetracycline-driven reverse transactivator (rtTA) (Gossen, D., et al. (1995), Science 268: 1766-1769) and hybrid transcriptional repressor Tet-KRAB (Deuschle, U., et al., (1995) Mol. Cell. Biol. 15: 1907-1914) modified to prevent the formation of an rtTA / Tet KRAB dimer (Freundlieb, S., et al., J. Gene Med. 1999; 1: 1-13).

Preferably The use of a vector that is a single tetracycline response element in the form of bidirectional Contains expression, provided basic activity by the TetR KRAB repressor is strictly repressed. In the absence of doxycycline, it binds TetR KRAB repressor strongly attached to the tetracycline response element and Ensures a drastic reduction in basic promoter activity. In the presence of doxycycline, transcription is triggered by detachment of the Tet-KRAB repressor and the binding of the rtTA activator activated to the promoter.

alternative The system can be based on a reverse silencer obtained by Fusion of the KRAB domain, which for the transcriptional repression is responsible, and the DNA-binding domain of rtTA in combination with the direct transactivator tTA. In this second Version of the regulatory system finds the transcriptional repression by culturing the cells in the absence of tetracycline.

Other Regulation systems are described in the art, e.g. those based on the use of ligands other than tetracycline, such as. RU486 (Wang, K.E., et al. (1994), PNAS, 91: 8180-8184), Ecdyson (No, D., et al. (1996), PNAS 93: 3346-3351), rapamycin (Spencer, D.A. M., et al. (1993), Science 262: 1019-1024), and their use can be easily adapted to the present invention. For the purpose Any gene expression regulator may be used in the present invention be used as long as it ensures adequate regulation and by the use of factors which are in the pharmacological Practice are acceptable, is inducible.

The presence of two DNA isolator di meren improves the stability of the second genetic unit, which allows its inducibility. These DNA elements may be derived from the chicken β-globin locus and are known to protect a transcriptional unit from constitutive inactivation due to DNA methylation and negative or positive influences which are due to the insertion site in the host chromosome.

Further can the cells of the present invention, a second genetic unit contained in a stable form, the non-structural regions, which is deleted in the ad genome of the first genetic unit present in the shuttle vector or inactivated in any way, functional with Sequences, preferably genomic sequences, which are inducible Regulate expression, linked are.

The second genetic unit may be in one or more vector (s) be contained and by transfection techniques, microinjection etc., into which cells are inserted.

A Selection markers, such as e.g. a G418 resistance gene, hygromycin B resistance gene, puromycin resistance gene, Bleomycin resistance gene is generally in both units and can be used to select cell lines that are stable or both the units maintained, being used in this The latter case is assumed either by a cell that has a contains the units, or cells that do not contain any of the units, in any case according to well-known techniques in this field.

The obtained after transformation with the second genetic unit Cells are characterized by the ability to express the information contained therein Non-structural areas in a strictly controlled manner. in the Case of regulation of non-structural areas using of the Tet system the addition of tetracycline to the culture medium for derepression of the Tet promoter mediated by tetracycline-induced dissociation of tTS from the TetR DNA binding site, followed by a complete transcriptional Activation due to rtTA binding.

Especially For example, this cell line comprises one or more in a stable form Vector (s) comprising the for E1 and E4 or E1, E2 and E4 coding genes which are the ad areas, that of the "ad / EBV shuttle vector" of a human ad virus like described above can be complemented. specially For example, the viral genes are those of the Ad5 serotype and the expression regulation system based on the use of both the tetracycline-driven reverse transactivator (rtTA) as well as the hybrid transcriptional Tet-KRAB repressor as disclosed above. However, you can genetic sequences from any other member of the Adenoviridae family derived are used. In their preferred embodiments this cell line is preferably the cell line A549 (human lung carcinoma, ATCC CLL 185) as described in the prior art other cell lines that allow adenovirus replication, in of the present invention also contemplated.

In In any of the cases described above, the derivative of the Helper cell line with a eukaryotic cell line, preferably a mammalian cell line, preferably of human origin, which is the first genetic unit in a stable episomal form as a vector (designated as a "shuttle plasmid") as disclosed above carried out.

In their preferred embodiments this cell line is preferably the cell line A549 (human lung carcinoma, ATCC CLL 185) as described in the prior art, but others Cell lines of human origin or not are from the scope of as long as they support adenovirus replication allow.

• a') Kultivierung der vorgenannten Helferzelllinie unter Bedingungen, welche dazu geeignet sind, die Expression von Genen, welche von den im Inneren der Zelle enthaltenen Adenovirus-Bereichen codiert werden, zu verringern, wobei die Bedingungen die im Stand der Technik für jeden hier beschriebenen Promotor bekannten Bedingungen sind, wie z.B. die in den Beispielen beschriebenen Bedingungen. Wenn das Tet-System für die transkriptionale Kontrolle verwendet wird, wird die Zelllinie in Abwesenheit von Tetracyclin kultiviert werden;
• b') Insertion in die Helferzelllinie der genomischen DNA für ein helfer-abhängiges Adenovirus durch Vektor-DNA-Transfektion oder Infektion der Zelle wie beschrieben (Parks, R. J., L. Chen, M. Anton, U. Sankar, M. A. Rudnicki und F. L. Graham (1996), Proc. Natl. Acad. Sci. USA 93:13656-13570).
• c') Induktion der Expression der Gene, welche von den im Inneren der Zelle enthaltenen Adenovirus-Bereichen codiert werden. Im Falle der tet-kontrollierten Transkription durch Zugabe von Doxycyclin zu dem Kulturmedium.

In a further aspect of the present invention, the helper cell lines thus obtained have been used in a process for the production of adenovirus-derived and helper-dependent vectors characterized by the following steps:

• a ') cultivating the aforementioned helper cell line under conditions suitable for reducing the expression of genes encoded by the adenovirus regions contained within the cell, the conditions being those known in the art for each promoter described herein known conditions, such as the conditions described in the examples. When the Tet system is used for transcriptional control, the cell line will be cultured in the absence of tetracycline;
• b ') Insertion into helper cell line of genomic DNA for helper-dependent adenovirus by vector DNA transfection or cell infection as described (Parks, RJ, L. Chen, M. Anton, U. Sankar, MA Rudnicki and FL Graham (1996), Proc Natl Acad Sci., USA 93: 13656-13570).
• c ') Induction of the expression of the genes which are encoded by the adenovirus regions contained inside the cell. In the case of tet-controlled transcription by adding doxycycline to the culture medium.

A large-scale production of a completely deleted HD adenovirus vector is achieved by serial vectoring as previously described for first generation vectors (Hitt, MM, et al., 1995, Meth. Mol. Genet., 7, 13-30).

specially this method is used for the production of helper-dependent adenovirus vectors, which for expression of polypeptides of therapeutic interest encoding genes preferably for use in gene therapy for use in human gene therapy.

So far A general description of the present invention has been given. A more detailed description of some specific embodiments this is given below with the help of the following examples, which a better understanding their goals, characteristics, advantages and embodiments should give.

Example 1: Construction an E1 and E4 expression vector

All Plasmids can using standard recombinant DNA techniques.

Of the E1 region of adenovirus 5 can be amplified by PCR using the viral genomic DNA can be obtained as a substrate and can be found in the plasmid pBI (Baron, U., et al. (1995), Nucleic Acids Res., 23 (17) 3605-3606) containing a bidirectional promoter, the flanked by a single tetracycline response element (TRE) of two minimal cytomegalovirus (CMV) promoters (Clontech), exists between the NotI and SalI restriction sites become what the plasmid pBI.E1 gives. Then pBI.E1 can be inserted the DNA, which for encodes the adenovirus E4 region obtained by PCR between the restriction sites MluI and NheI, thus construct pBI.E1 / E4 to obtain.

Example 2: Construction an E1 / E4 expression vector

All Plasmids can using standard recombinant DNA techniques.

The E4 ORF6 gene DNA was amplified by PCR using the oligonucleotides 5'-TTATACGCGTGCCACCATGACTACGTCCG-3 'and 5'-TTATGCTAGCGCGAAGGAGAAGTCCACG-3' and pFG140 containing viral Ad5 genomic DNA as a substrate. Amplified DNA was transformed into the plasmid pBI (Clontech) (Baron, U., et al. (1995), Nucleic Acids Res., 23 (17) 3605-3606), which contains a bidirectional promoter consisting of a single tetracycline response. Element (TRE) flanked by two minimal cytomegalovirus (CMV) promoters, inserted between the restriction sites MluI and NheI, which gives the plasmid pBI.E4. pBI.E4 was prepared by inserting the DNA coding for the adenovirus E1 region obtained by PCR using the oligonucleotides 5'-ATGCGCGGGCGCTGAGTTCCTCAAGAGG-3 'and 5'-ATGCGTCGACCAGTACCTCAATCTGTATCTTC-3' between the NotI and SalI restriction sites, wherein finally the construct pBI.E1 / E4 was obtained ( 1 ).

expression vectors for E2a and E2b genes were constructed following the same strategy, the plasmid pFG140 was used to prepare DNA binding protein gene DNA and pVACpoI and pVACpTP as templates for amplification the cDNA of polymerase and preterminal Protein (pTp). DBP DNA was inserted into pTRE (Clontech) between the restriction sites EcoRI and XbaI cloned. Ad polymerase and pTP were in the same Vector cloned or combined into the bi-directional pBI Promoter under the transcriptional control of the Tet operator.

Example 3: Construction a vector, the tetracycline-driven transcriptional regulators expressed

The Tet control system allows regulation of gene activities over a broad range of mammalian cell sizes. A transcriptional silencer (tTS) was recently developed by fusing the DNA-binding domain of the tetracycline repressor to the silencer domain of Kid-1 (Freundlieb, S., et al., J. Gene Med. 1999; 1: 1-13 ). tTS and rtTA were combined in a single expression vector as follows. An EcoRI-ClaI DNA fragment containing the Tet silencer was isolated from the plasmid pUHS6-1 and inserted downstream of the IRES sequence into the vector pIRES-Neo (Clontech) replacing the Neo gene. The new vector pIRES-tTS was modified with the insertion of the rtTA gene into the unique EcoRV restriction site downstream of the human cytomegalovirus IE promoter, which yielded pIREStTS / rtTA. To introduce a selection marker for the isolation of cell clones which stably expressed Tet proteins, a puromycin resistance expression cassette obtained from the vector pPUR (Clontech) was inserted into the XhoI site of pIREStTS / rtTA, which gave pIREStTS / rtTApuro ( 2 ).

Example 4: Construction of the shuttle plasmid

A 32462 bp DNA fragment containing the entire adenovirus 5 genome in which the E1 region and the packaging signal are deleted can be obtained by digesting the plasmid pBHG10 (Bett et al., Proc. Natl. Acad Sci : 8802-8806, 1994) with the restriction enzymes XbaI and ClaI are obtained. The resulting fragment can be inserted into the vector pCEP4 (Invitrogen) containing the replication origin of EBV Ori P and the gene for the viral transactivator EBNA-1, between the restriction sites SspBI and BamHI, to thereby obtain the plasmid pSC. Then, the E4 region of the adenovirus genome can be deleted, eliminating the DNA fragment contained between nucleotides 32810 and 35620, and thus generating the plasmid pSCΔE4. Subsequently, pSCΔE4 modifications can be introduced to optimize the vector properties. For example, re-insertion of sequences from the AdS-Wt-E3 region deleted in the context of pBHG10 into the viral genome may increase expression levels of the gene encoding the viral fiber. In addition, a DNA fragment including tetracycline repressor multiple binding sites (tet-O) may be inserted upstream of the E2 region transcriptional starts to further attenuate the residual expression of the adenovirus genome as described by Rittner et al. (Rittner, K., et al., (1997) J. Virol 71: 3307-3311). In this version of the shuttle plasmid pSCΔE4 its expression is further attenuated by Tet-KRAB, which binds to the E2 region in the absence of tetracycline.

Example 5: Construction of the shuttle plasmid

A: Subcloning and modification of the E4 range of Ad5

One DNA fragment containing the entire adenovirus 5-E4 region obtained by cleaving the plasmid pBHG10 (Bett et al., Proc. Natl. Acad. Sci. 91: 8802-8806; 1994) with the restriction enzymes SpeI and ClaI. The isolated fragment was inserted into the vector pBluescript (Stratagene) ligated between the restriction sites SpeI and ClaI, which is the Plasmid pBSE4 resulted. Then pBSE4 was inserted by insertion of an eptamer of DNA binding sites for the Tet repressor into the unique PacI restriction site which resulted in pBSE4-ept. The Tet-Eptamer DNA was analyzed by PCR using the oligonucleotides 5'-CTGATTAATTAAATAGGCGTATCACGAGGCC-3 'and 5'-CTGACGATCGCGTACACGCCTACTC-3' and the plasmid pUHD10.3 amplified as DNA template. The Tet binding site was into the PacI restriction site of pBSE4 just upstream of the E2 promoter cloned. The ultimate goal was to reduce background expression of the E2 promoter using the silencer effect of the tetracycline-controlled transcriptional silencers as described by Rittner (Rittner, K., et al. (1997) J. Virol. 71: 3307-3311). The Ad5 E4 region present in PBSE4-ept was then eliminated by digestion with the restriction enzymes MfeI and ClaI, the vector DNA was gel purified and treated with a Tk-hygromycin B-resistance expression cassette DNA obtained by PCR with the Oligonucleotides 5'-AGTGCACAATTGATTTAAATAATCCGCGCGGTGG-3 'and 5'-TGCAATCGATCAACGCGGGCATCC-3' using of pCEP-4 plasmid DNA as template, which yielded pBSΔE4. The Adenovirus ITRs in head-to-tail configuration were then used PCR using the oligonucleotides 5'-TCGAATCGATACGCGAACCTACGC-3 'and 5'-TCGACGTGTCGACTTCGAAGCGCACACCAAAAACGTC-3' and pFG140 (Microbix) plasmid DNA as template amplified. Ad-ITRs were added to the unique NruI site from pBSΔE4 cloned, which gave pBSΔE4J.

B: insertion of EBV-OriP in pLBG40

One DNA fragment containing EBV OriP was carried out from the vector pCEP4 MfeI digestion isolated and in PI 28 (N.E. Biolabs) digested with the restriction enzyme EcoRI, subcloned. The resulting plasmid plit-OriP was then digested with XbaI to a 2476 bp DNA fragment containing the EBV OriP which is in the only once occurring XbaI site of the plasmid pLBG40 (Recchia, A., et al., 1999, Proc. Natl. Acad. Sci. 96: 2615-2620) which gave pLBG-OriP.

C: Construct of Ad / EBV shuttle plasmids

A shuttle plasmid pSA-1 ( 2 ) carrying a copy of the Ad5 genome deleted E1, E3, E4 and the packaging signal was constructed by replacing the DNA fragment of pLBG-OriP between the restriction sites SfuI and PacI by the PacI-ClaI DNA Fragment obtained from pBSΔE4J.

pSA-1 was further modified by insertion of the EBNA-1 gene as follows. pREP10 (Invitrogen), a plasmid containing both elements of the EBV origin of EBV replication, EBNA-1 and OriP, was digested with XbaI and NheI to isolate a 429 bp DNA fragment containing SV40 polyA. Plasmid DNA was completely filled in, gel purified and ligated to generate pREP11. pREP11 was then digested with EcoRI and XhoI to release a DNA fragment containing both the EBNA-1 gene and the OriP. This DNA fragment was subcloned into pLitmus28 digested with EcoRI and XhoI to obtain pREP12. pREP12 was digested with EcoRI and MluI, gel purified, and ligated to the 3627 bp EcoRI-MluI DNA fragment isolated from pCEP4 to generate pREP13. pREP13 was digested with XbaI and NheI to form a Release DNA fragment with XbaI compatible ends containing EBNA-1 and OriP. Finally, this fragment was cloned into pSA-1 digested with XbaI to generate pSA-2.

Example 6: Ad / EBV Shuttle Plasmid Modifications

The Ad / EBV shuttle plasmids (pSA-1 and pSA-2) were further modified by deleting a DNA sequence corresponding to the DNA binding protein (DBP) gene (nucleotides 22443-24032 the Ad5 sequence) corresponded. The deletion was by homologous recombination in E. coli according to that described by A.F. Stuart and coworkers Methods (Zhang et al., Nat. Genet. 1998; 20: 123-128). A DNA fragment containing the Tn5 kanamycin resistance gene (neo), flanked by DNA sequences was amplified by PCR with the oligonucleotides 5'-GCGGTTAGGCTGTCCTTCTTCTCGACTGACTCCATGATCTTTTTCTGCCTATAGGAGAAG GAATCCCGGCGGATTTGTCCTACTCAGGAGAGCG-3 'and 5'-AAATGCTTTATTTGTACACTCTCGGGGATTATTTACCCCCACCCTTGCCGTCTGCGCC GTTCTGCAAACCCTATGCTACTCCGTCG-3 ', consisting of about 60 bp homology to the DBP gene, and to the 3 'ends PCR primers for amplifying the neo gene using pGKfrt as Die received. Linear DNA containing the neo gene has been included in Recombination experiments were used to delete the DBP gene from Ad / EBV shuttle plasmids. The same procedure was used to construct Ad / EBV shuttle plasmids, which express no ad polymerase gene and preterminal protein. The sequence of the oligonucleotides used to delete the polymerase gene was 5'-ACGGCCTGGTAGGCGCAGCATCCCTTTTCTACGGGTAGCGCGTATGCCTGCGCGGCCT TCCGGTCTGCAAACCCTATGCTACTCCGTCG-3 'and 5'-AGACCTATACTTGGATGGGGGCCTTTGGGAAGCAGCTCGTGCCCTTCATGCTGGTCAT GTCCCGGCGGATTTGTCCTACTCAGGAGAGCG-3 '. Two pairs of oligonucleotides were added deletion of the two regions within the main excretory pTP used: 5'-CCGCCTCCCGGTGCGCCGTCGTCGCCGCCGTGTCCCCCCTCCCCCACCGTCCCGGCG GATTTGTCCTACTCAGGAGAGCG-3 'and 5'-GATCTCCGCGTCCGGCTCGCTCCACGGTGGCGGCGAGGTCGTTGGAAATGCGTCTGC AAACCCTATGCTACTCCGTCG-3 ', 5'-TCGACAGAAGCACCATGTCCTTGGGTCCGGCCTGCTGAATGCGCAGGCGGTCTGCAAA CCCTATGCTACTCCGTCG-3 'and 5'-TCGCCCCCGGAGCCCCGGCCACCCTACGCTGGCCCCTCTACCGCCAGCCGCTCCCGG CGGATTTGTCCTACTCAGGAGAGCG-3 '.

The same procedure may apply to other areas of genomic adenovirus DNA be applied for the achievement of a reduction in cytotoxic effects caused by expression of viral genes in the infected cell become, are of importance.

Example 7: Assessment of E1 / E4 expression

to Determination of the regulation of E1 gene expression in cells containing Tet proteins HeLa cells were seeded on 6-well plates and cultured with pBI.E1 / E4 transfected in combination with pIREStTS / rtTApuro. The Experiment was done in duplicate with and without doxycycline. 48 h after transfection HeLa cells were harvested and cell lysates using Western blots using a monoclonal antibody, the directed against E1 proteins was, analyzed. 293 cells constitutively expressing the E1 region, were used as a positive control.

Leak expression of E1 proteins was detectable in cells transfected with pBI.E1 / E4 only. As expected, strong expression of E1 proteins induced by doxycycline was observed in the presence of Tet regulatory factors, whereas expression was undetectable in the absence of the drug. This result demonstrates that the expression of Tet-Silencer actively represses gene activity and thus eliminates background expression. Concomitant expression of tTS and rtTA did not affect gene induction via rtTA in the presence of doxycycline. This result was confirmed by a second experiment in which HeLa cells were infected with a ΔE1 Ad vector expressing β-galactosidase and then transfected with pBI.E1 / E4 and pIREStTS / rtTApuro. 48 h after transfection, HeLa cells were harvested and a lysate was obtained by disrupting the cells by freezing and thawing. Monolayers of A549 cells were incubated with an aliquot of the cell lysate and after 24 hours β-gal activity was detected as described (Parks, RJ, et al., 1996, Proc Natl Acad Sci., 91: 8802-8806) , Since HeLa cells are not completely permissive for replication of first generation vectors, no viral progeny were detected in the cells that were only infected, whereas the ΔE1 vector was detected in cells spiked with both vectors (pBI.E1 / E4 and pIREStTS / rtTApuro) was fully complemented in the presence of doxycycline, as demonstrated by the detection of lacZ-transducing particles in the cell lysate. A low β-galactosidase transduction titer was detected after transfection with pBI.E1 / E4 as a result of Tet promoter basal activity. In contrast, no transducing particles were detected in cells maintained in the absence of doxycycline after cotransfection of pBI.E1 / E4 and pIREStTS / rtTApuro and infection with Ad-ΔE1-βgal. This result confirms that modulation of E1 gene expression can be obtained by combining Tet-Silencer and rtTA activity. In addition, tTS expression represses E1 production below the level required to detect viral replication of the ΔE1 ad vector.

Example 8: Complementation Function of Ad / EBV shuttle plasmids

To the Testing of Ad helper function of Ad / EBV plasmids were the plasmids in A549EBNA and 293EBNA cell lines in combination with the helper-dependent Ad plasmid C4E1E4gfp cotransfected. This vector contains E1 and E4 adenovirus regions and an expression cassette for green fluorescent protein (GFP). If both plasmids were introduced into the same cell, the expression of the E1 and E4 genes activated the Ad / EBV plasmid, which replicates both vectors, but only the packaging allowed by C4E1 / E4gfp DNA. Two parallel experiments were performed Use of both cell lines performed. 72 hours after transfection was episomal DNA from a tissue culture dish according to the Hirt method extracted. Transfected cells were harvested from the second dish and broken up by freezing and thawing to give a crude cell lysate to obtain.

episomal DNA was digested with the restriction enzymes HindIII and DpnI and analyzed by Southern blots. Filters were probed with a hygromycin B DNA probe hybridizes which for the Ad / EBV plasmid was specific, and then, after detachment, with a second DNA probe derived from C4E1E4gfp, both Plasmids recognize, rehybridized. Parallel to this was a monolayer of 293 cells with aliquots of cell lysate incubated to produce to detect gfp-transducing particles. The results demonstrated that Ad / EBV plasmids are circular in shape and replicate in linear form when both Ad (E1 / E4) and viral EBV (EBNA-1) transactivator. Furthermore led activation of the ad transcription program to C4E1E4gfp DNA replication and packaging in mature virions, as demonstrated by the observation of fluorescent GFP expressing cells in the 293 monolayer that interact with the cell lysate was incubated.

Example 9: Construction a cell line expressing regulatory proteins

A549 cells (Human lung carcinoma, ATCC CLL 185), which control both the tetracycline reverse transactivator (Gossen, D., et al. (1995), Science 268: 1766-1769) as well as the hybrid transcriptional tet-KRAB repressor (Deuschle, U., et al. (1995), Mol.

Cell. Biol. 15: 1907-1914) can be expressed by co-transfection of the both plasmids pTet-On (Clontech) and pTetKRAB are obtained. Subsequently, the cell clones obtained by selection with the antibiotic G-418 be tested by using any vector in which a reporter gene is inserted under the control of the Tet operator, expression to determine both transregulating proteins.

One Adenovirus vector of the first generation was constructed by into the PacI site of the plasmid pLBG40 an expression cassette, which contained the luciferase gene placed under the control of the Tet operator, was inserted, whereby the plasmid pLBGluc was obtained.

This Plasmid comprises the entire adenovirus genome, in which the E1 and E3 areas are deleted, and is therefore infectious if it is inserted into 293 cells by transfection. The virus (AdLBGluc), that in the plaques, which occurs about 10 days after transfection into 293 cells, which were cultivated in monolayer, appeared, is contained expanded, titrated and assayed for luciferase enzyme expression subjected.

Approximately 10 ⁴ cells of each clone, obtained by resistance to G418, can be seeded in 24-well plates and infected with AdLBGluc virus at an MOI of 20. The same number of cells were seeded as a duplicate on a second plate and cultured in the presence of doxycycline prior to infection. 48 h after infection, the cells were harvested and lysed. The expression levels of the luciferase gene were determined using the natural substrate luciferin. The clones in which the best relationship between the activation of luciferase expression in the presence of doxycycline and the basal level in the absence of ligand was observed were selected and expanded. A clone prepared according to this method has been expanded to further define characteristic properties of growth, stability and expression levels of regulatory proteins. Therefore, suitable banks of frozen cells were prepared to ensure the maintenance of a cell line expressing the regulatory proteins of the transcription rtTA and Tet-KRAB.

Example 10: Construction the E1 / E4-inducible cell line

The plasmid pBI.E1 / E4 (see Example 1) can be inserted into the cells of the previously selected clone together with a plasmid expressing resistance to the antibiotic puromycin, be transfected. The cells are selectable by growth on a medium containing the antibiotic and are assayed for their expression of adenovirus early proteins under the control of tetracycline. For this purpose, a second generation adenovirus vector deleted of both the E1 and E4 regions can be constructed as described in the literature (Brough, DE, et al., (1996) J. Virol. 70: 6497-6501). The cell clones obtained by insertion of the transcriptional unit E1 / E4 into the cell can be selected on the basis of their ability to complement the helper-dependent defective adenovirus and thus permit its replication. Puromycin-resistant clones can be seeded in 24-well plates as a duplicate. Subsequently, the cells can be infected with the ΔE1 / E4 virus and then cultured with and without addition of doxycycline to the culture medium. In the presence of doxycycline, E1 and E4 transcriptional activation may render cells permissive for viral replication, thus demonstrating the cytopathic effect associated therewith. The clones in which the AdΔE1 / E4 vector can replicate only in the presence of doxycycline can be expanded and further characterized to detect persistent production of the defective virus.

Example 11: Construction an EBNA-1-expressing A549 cell line

A A549 cell line expressing an EBNA-1 gene was characterized by stable Transfection of the vector pEB (Ramage, A.D., et al., 1997, Gene 197: 83-89) generated. pEB was linearized by MluI digestion and into A549 transfected using the reagent Fugene 6 (Boehringer). 72 h after transfection, A549 cells were in a 1:20 ratio in selective medium containing 600 μg / ml G418. 10 resistant Clones were expanded and for episomal replication of the plasmid pCEP-EBNAde1, containing the EBV-Ori-P and a hygromycin resistance gene expression cassette, tested.

Example 12: Construction the helper cell line

The Shuttle plasmid pSCΔE4 can be induced by transfection techniques into the cell line which is the E1 / E4 inducible transcriptional unit, be inserted. The resistant ones Cell clones obtained by culturing the transfected cells in the presence of the antibiotic hygromycin B can be expanded and further in terms of their capacity for inducible expression the adenovirus genes and to maintain the propagation of a helper dependent Ad vectors are characterized. First, the inducible expression of the adenovirus genome be, with the mediated cytopathic effect by culturing the cells is detected in the presence of tetracycline. The production The adenovirus structural proteins can be quantified by Western blotting and immunoprecipitation techniques with specific antibodies, as already described in the literature. The capacity of cell clones, which the episome pSCΔE4 lock in, a replication of helper-dependent Vectors can be constructed using different vectors, which the reporter genes responsible for the green Fluorescent protein (GFP) or for the β-galactosidase or encode any other gene with a readily detectable activity, to be examined. The cells can be infected with a different MOI of the helper-dependent virus, then harvested, if the cytopathic effect is evident, after tetracycline-induced expression of the adenovirus genome. The virus yield can using the cellular Lysates as described by Parks in PNAS 1996. The determination of the ratio between the number of transducing viral particles formed, which infect the packaging cell lines and those which are in the viral inoculum used, an estimate of the Virus production efficiency in the various clones obtained enable.

Example 13: Construction of cell lines containing Ad / EBV shuttle vector

A. A549 EBNA clones Ad / EBV shuttle vector included

Ad / EBV shuttle plasmid was transferred to the A549 EBNA cell line using the transfection reagent Fugene-6 (Boehringer) or the calcium phosphate method. Ad / EBV plasmid was used alone or in combination with pIREStTS / rtTApuro or pUHS6-1 transfected. Cotransfection of the Ad / EBV shuttle vector with Plasmids containing the tet-silencer should express the occurrence of ad gene leak expression by Further reduce repression of the E2 promoter. A549 EBNA cells were transferred to selective medium containing 400 μg / ml G-418 and 200 μg / ml hygromycin-B contained 48-72 h after transfection. Isolated hygromycin B resistant Clones were transferred to 24-well plates and expanded.

To test A549 Ad / EBV clones for the ability to complement and propagate a helper-dependent Ad vector, a novel plasmid C4E1E4gfp was constructed. This helper-dependent plasmid contains E1 and E4 adenovirus regions as well as a green fluorescent protein expression cassette. The HDC4E1E4gfp vector was released and amplified using 293CRE4 cells as described (Parks, RJ, et al., 1996, Proc. Natl. Acad. Sci. 93: 13565-13570). A purified preparation of HDC4E1E4gfp was used to infect A549 Ad / EBV clones at a low multiplicity of infection.

Clones after Ad / EBV cotransfection with pIREStTS / rtTApuro or pUHS6-1 were duplicate with or without 1 μg / ml doxycycline tested. Expression of E1 and E4 genes allowed activation of the ad transcription program only in clones that are intact Contained copy of the Ad / EBV shuttle plasmid. The result was the Induction of a cytopathic effect (cpe) and HD vector propagation.

B. Construction of various Cell lines containing the Ad / EBV shuttle vector II

pSA-2 incorporates an EBNA-1 expression cassette and is thus in the Able, in any cell line, in terms of episomal maintenance is permissive, mediated by a latent EBV origin of replication to replicate. He was then identified for newer, by A549EBNA different cell lines with improved biological properties used. A number of different cell lines were transfected and hygromycin B resistant Clones were isolated and examined as for the clones, obtained from A549EBNA cells been described.

Example 14: Isolation of a cell clone that regulates E1 and E4 proteins in a tet-regulated manner Way expressed

A549EBNA cells which is both the tetracycline-controlled reverse transactivator (Gossen, D., et al. (1995), Science 268: 1766-1769) as well as the transcriptional tTS silencer (Deuschle, U., et al., (1995), Mol. Cell. Biol. 15: 1907-1914; Freundlieb, S., et al., J. Gene Med. 1999; 1: 1-13) were expressed by Transfection of pIREStTS / rtTApuro received. Subsequently were the cell clones obtained by selection with the antibiotic puromycin tested the expression of the two transregulating proteins by using any vector in which a reporter gene is under the control of the Tet operator is installed to determine.

One Adenovirus vector of the first generation was constructed by Insertion of an expression cassette containing the luciferase gene contains the control provided by the Tet operator, which by PvuII digestion from pABS-Tetluc into the XbaI site of plasmid pLBG40, thereby the plasmid pLBGTetluc was obtained.

This Plasmid comprises the entire adenovirus genome, in which the E1 and E3 areas are deleted, and is therefore infectious in transfection in 293 cells. Isolated plaques were 10 days after 293 transfection visible, noticeable. Three plaques were picked and by HindIII digestion after that by Graham, F.L., and coworkers, 1995, Methods in Molecular Genetics 7: 13-20, described procedures. All three Plaques showed the correct restriction pattern. A viral isolate was expanded, titrated and for luciferase enzyme expression subjected to an assay.

About 10 ⁴ cells of each clone, obtained by resistance to puromycin, were seeded as a duplicate in 24-well plates and infected with AdLBGluc virus at an MOI of 20. A plate was cultured in the presence of doxycycline prior to infection. The cells were harvested 48 hours after infection and lysed. The expression levels of the luciferase gene were determined using the natural substrate luciferin. The clones, in which the best ratio between the activation of luciferase expression in the presence of doxycycline and the basal level in the absence of ligand is observed, were selected and expanded. A clone prepared according to this procedure was expanded to further define characteristic properties of growth, stability and expression levels of regulatory proteins. Therefore, suitable banks of frozen cells were prepared to ensure the maintenance of a cell line expressing the regulatory proteins of the transcription rtTA and Tet-KRAB.

Example 15: Construction the E1 / E4-inducible cell line

The plasmid pBI.E1 / E4 (see Example 1) was transfected into the A549EBNA cells together with pIREStTS / rtTApuro. The cells were selected by growth on a medium containing the antibiotic and assayed for expression of adenovirus early proteins under the control of tetracycline. For this purpose, a second generation adenovirus vector, with both the E1 and E4 regions deleted (Krougliak, V., et al., 1995, Hum. Gene Ther., 6: 1575-1586), was deleted from FL Graham and used to screen for positive clones. The cell clones obtained by insertion of the E1 / E4 transcriptional unit into the cell were selected on the basis of their capacity to complement the helper-dependent defective adenovirus vector and thus enable its replication. Puromycin-resistant clones were seeded in 24-well plates as a duplicate. Then, the cells were infected with the ΔE1 / E4 virus and then cultured with or without addition of doxycycline to the culture medium. In the presence of doxycycline, the E1 and E4 transcriptional activation make cells permissive with respect to viral replication, thus demonstrating the mediated cytopathic effect. Viral titers were determined by PCR using the TaqMam method. The clones in which the AdΔE1 / E4 vector can replicate only in the presence of doxycycline were expanded and further characterized, whereby a permanent production of the defective virus was determined. The same procedure was followed to obtain A549EBNA clones expressing E2 genes under tetracycline control. The resulting clones were screened using Ad vectors in which the corresponding E2 genes were deleted.

Example 16: Construction the helper cell line and HD propagation

The Shuttle plasmid pSA-1 was prepared by standard transfection techniques inserted into the cell line containing the E1 / E4-inducible transcriptional unit and Tet proteins included. Several resistant clones were obtained by dividing the cells 48 hours after transfection and cultivation in a selective medium that also contains hygromycin B in a concentration of 200 μg / ml contained. Resistant clones have been expanded and in terms of their assets inducible expression of the adenovirus genes and for maintenance the propagation of a helper-dependent Characterized further Ad vector. Initially, the inducible Expression of the adenovirus genome checked using the mediated cytopathic Effect of culturing the cells in the presence of tetracycline was determined. The induction of adenovirus structural protein production was quantitated by western blots and immunoprecipitation techniques with specific antibodies. The capacity of the cell clones that included the episome pSA-1 the replication of helper-dependent Vectors were examined using different vectors, which the reporter genes coding for the green fluorescent protein (GFP) or for the β-galactosidase or any other gene with readily detectable activity. The cells were challenged with a different MOI of helper-dependent virus infected, then harvested when the cytopathic effect becomes apparent was after tetracycline-induced expression of the adenovirus genome. The Virus yield became infection by using the cell lysate of a 293 cell monolayer. The determination of the ratio between the number of formed transducing viral particles, which infected the packaging cell lines, and those present in the viral inoculum was used to assess virus production efficiency used in the various clones obtained.

the same Procedure was followed to modify the packaging cell lines with the modified ones Versions of the Ad / EBV shuttle plasmid combining different Deletions earlier Genes, including E2 genes, to obtain.

REFERENCES

• - Baron, U., et al. (1995), Nucleic Acids Res., 23 (17): 3605-3606
• - Brough, D.E., et al. (1996) J. Virol. 70: 6497-6501
• - Bed et al. (1994), Proc. Natl. Acad. Sci. 91: 8802-8806
• - Calos, M.P. (1996), Trends Genet 12: 463-466
• - Calos, M.P. (1998), Proc. Natl. Acad. Sci. USA 95: 4084
• - Deuschle, U., et al. (1995), Mol. Cell. Biol. 15: 1907-1914
• - Englehardt et al. (1994), Proc. Natl. Acad. Sci. 91: 6196-6200
• - friend love, S., et al. (1999), J. Gene Med. 1: 1-13
• - gutters, D., et al. (1995), Science 268: 1766-1769
• - Hitt, M.M., et al. (1995), Meth. Mol. Genet. 7: 13-30
• - Hitt M.M., et al. (1997), Advances in Pharmacol. 40: 137-206
• - Horvitz, "Adenoviridae and their replication "in Virology, Field and Knipe, ed. Raven Press, NY; 1990; Pages 1679-1740
• - Kozarsky et al. (1994) J. Biol. Chem. 269: 1-8
• - Krougliak, V., et al. (1995) Hum. Gene Ther. 6: 1575-1586
• - No, D., et al. (1996), PNAS 93: 3346-3351
• - Parks, R.J., L. Chen, M. Anton, U. Sankar, M.A. Rudnicki, and F.L. Graham (1996), Proc. Natl. Acad. Sci. USA 93: 13656-13570
• - Sambrook J., Fritsch, E.F., and Maniatis, T. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
• - Spencer, D.M., et al. (1993), Science 262: 1019-1024
• - Wang, K.E., et al. (1994), PNAS, 91: 8180-8184
• - Yang et al. (1994), Proc. Natl. Acad. Sci. 91: 4407-4411

Claims (25)

Cells for the production of helper-dependent adenovirus vectors which comprise at least the following genetic units: a first genetic unit comprising a defective adenovirus genome in which the inverted terminal repeats are in a head-to-tail configuration, the Packaging signal is inactivated and at least one of the non-structural areas is inactivated; A second genetic unit comprising at least one inducible promoter and at least one of the regions inactivated in the first genetic unit, the regions being under the control of the inducible promoter; - whereby after the activation of the inducible promoter of the second genetic unit and the Infection of the cells with the helper-dependent adenovirus vectors enable the first genetic unit and the second genetic unit to produce the helper-dependent adenovirus vectors in the cells in the absence of helper virus. Cells according to claim 1, wherein the first genetic Unit is integrated in the genome of the cells and at both ends having inverted terminal repeats in a head-to-tail configuration. Cells according to claim 1, wherein the first genetic Unit is included in an episomal unit, the one Element includes the replication of the episomal unit in a low copy number. Cells according to claim 3, wherein the element which the replication of the episomal unit allows the replication origin a virus is. Cells according to claim 4, wherein in the episomal unit furthermore, the gene which is responsible for includes an activation factor of the replication origin, included is. Cells according to claim 4, wherein the gene coding for a Activation factor of the replication origin encoded in the genome is integrated. Cells according to any one of claims 4 to 6, wherein the virus the Epstein-Barr virus is, the replication origin is OriP and the activation factor is EBNA-1 is. Cells according to any one of claims 1 to 7, wherein the packaging signal of the defective adenovirus genome of the first genetic unit full or partial deletion is inactivated. Cells according to any one of claims 1 to 8, wherein the non-structural Regions of the defective adenovirus genome of the first genetic unit through complete or partial deletion are inactivated. Cells according to any one of claims 1 to 9, wherein the inactivated areas of the first genetic unit from the group consisting of E1, E2A, E2B and E4 are selected. Cells according to claim 10, wherein the regions E1 and E4 are. Cells according to claim 10, wherein the regions E1, E4 and E2A are. Cells according to claim 10, wherein the regions E1, E4 and E2B polymerase are. Cells according to claim 10, wherein the regions E1, E4 and präterminales E2B protein (PTP). Cells according to any one of claims 1 to 14, the viral regions of the first genetic unit being functional with are associated with at least one regulatory element, which allows strict control of the expression of these areas. Cells according to any one of claims 1 to 15, where the promoter on the second genetic unit is the tetracycline operator is. Cells according to any one of claims 1 to 16, wherein the viral regions in the second genetic unit are functional with Elements are linked, which regulate the expression of these regions. Cells according to any one of claims 1 to 17, being the defective adenovirus genome the first unit completely or partially from the genome of a human adenovirus. Cells according to claim 18, wherein the defective adenovirus genome the first genetic unit completely or partially from the Genome of at least one of the human adenoviruses Ad2 and Ad5 exists. Cells according to any one of claims 1 to 19, wherein the viral regions of the second genetic entity are complete or consist partly of viral regions of a human adenovirus. Cells according to claim 20, wherein the viral regions of the second genetic unit wholly or partly of viral Areas of at least one of the human adenoviruses Ad2 and Ad5 consist. Cells according to any one of claims 1 to 21, wherein the cells are mammalian cells are. Cells according to claim 22, wherein the mammalian cells are human cells. A composition comprising the cell of claim 1 and a vehicle or carrier, wherein the composition is free of helper viruses. A process for the production of helper-dependent adenovirus vectors, comprising: (a) culturing cells according to any one of claims 1 to 23; (b) introduction of genomic DNA of the helper-dependent adenovirus vectors into the cells; and (c) activating the at least one inducible Promoter of the second genetic unit.